tbuckley/cabot-search huggingface dataset:
INNV-06. DETECTION OF MUTANT P53 POSITIVE GLIOBLASTOMA CELLS IN CEREBROSPINAL FLUID (CSF) USING A MICROFLUIDIC BASED IMMUNOASSAY
Authors: Steven Hsiao, Tony Pircher, Nathan Sweed, Kaitlyn Armijo, Mikayla Marrin, Leslie Cho, Michael Dugan, Santosh Kesari
Journal: Neuro-Oncology (2022)

Abstract BACKGROUND Metastatic involvement of the CSF by non-CNS neoplasms surpasses that of primary brain tumors, although conventional glioblastoma (GBM) can occasionally be identified in the CSF. Here, we apply Biocept’s CNSide test to examine a patient CSF sample with GBM, verified with an antibody against mutant p53. The p53 pathway is deregulated in 84% of glioblastoma patients and point mutations lead to expression of mutant p53 protein. METHODS A CSF sample from 65-year-old male patient, with GBM diagnosed by MRI &amp; CT, and prior identified p53 mutation on R273C by NGS was collected into Biocept’s CSF collection tubes at Providence Saint John’s Health Center. CSF-cells were incubated, 2 days post collection, with a proprietary antibody cocktail, including anti-CD9, followed with a biotinylated secondary, which enables enrichment of capture antibody labeled cells in a streptavidin coated microfluidic device. Cells were fixed and permeabilized immunofluorescence was performed against mutant p53, CD45, fluorescently labeled streptavidin, for capture antibody detection, and DAPI. Microfluidic chips were scanned and analyzed on a Bioview system. The specificity of the mutant p53 antibody was verified using the same test on tumor cells with either wildtype p53 vs various p53 mutations. RESULTS CSF analysis by the CNSide platform detected 3 tumor cells / mL. Immunofluorescence confirmed strong expression of mutant p53 protein on about 1/3 of cells identified as tumor cells. Besides the patient R273C mutation, we demonstrated detection of P223L/V274F, E285K and Q331R mutations on tumor cell lines. DISCUSSION Our work suggests that a microfluidic based GBM capture test paired with appropriate biomarkers can be used as sensitive means to detect GBM cells in CSF and may be useful for diagnostics and treatment monitoring. Further, we demonstrated the utility of a mutant p53 antibody to identify tumor cells. Technical and clinical studies are needed to substantiate this hypothesis.
BIOM-60. APOLLO: RAMAN-BASED PATHOLOGY OF MALIGNANT GLIOMA
Authors: Adrian Lita, Joel Sjöberg, Stefan Filipescu, Orieta Celiku, Luigia Petre, Mark Gilbert, Houtan Noushmehr, Ion Petre, Mioara Larion
Journal: Neuro-Oncology (2022)

Abstract Methylation classification is an essential component for integrative diagnosis in glioma, however, the DNA methylation classification is not always available for all the samples. We hypothesized that Raman spectroscopy might be suitable to predict the glioma methylome, based upon its ability to create a molecular fingerprint of the tumor and would provide biological insights into the discriminatory features. Raman Spectroscopy was used for molecular fingerprinting of the regions of interest using 1mm2 FFPE tissue spots from 45 patient samples with LGm1 to LGm6 methylation subtypes. Spectral information was then used to train a convolutional neural network (CNN), capable of detecting the glioma methylation subtypes. 70 % of the dataset was used for model training while the remaining 30% for validation. We demonstrate that Raman spectroscopy can accurately and rapidly classify gliomas according to their methylation subtype from achieved FFPE samples, as a novel way to obtain classification. For each sample we ran Ward linkage clustering with a variable number of clusters (from 2 to 7), with the majority cluster corresponding to tumor spots and the others corresponding to (various types of) non-tumor spots. The average accuracy over all samples was 90:3%, the average precision was 99:6% and the average recall was 90:2%. We show that Raman spectroscopy together with artificial intelligence can predict the methylome of glioma samples and augment the ability to classify these tumors retrospectively. The non-destructive nature of this method and the ability to be applied on FFPE samples directly, allows the histopathologist to reuse of the same slide for subsequent staining and downstream analyses.
EXTH-51. HSV-PTENΑ TREATMENT TARGETS GLIOMA STEM CELLS TO REDUCE STEMNESS AND SENSITIZES GLIOBLASTOMA TO IRRADIATION
Authors: Matthew P Mullarkey, Upasana Sahu, Bangxing Hong, Balveen Kaur
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma is one of the most lethal and treatment-resistant tumors of the central nervous system. Despite maximal surgical and medical therapy, survival remains dismal with a median of 21 months. Despite advances in treatment, this has only led to modest survival benefit. A significant challenge in treatment of glioblastoma is targeting glioma stem cells (GSCs) which are a source of therapy resistance. Oncolytic viral (OV) therapy is a promising therapy for solid tumors that preferentially targets tumor cells for lysis and an anti-tumor immune response while sparing normal cells. Among all OVs, oncolytic Herpes Simplex Virus (oHSV) is substantially ahead in the clinic, with an oHSV T-VEC approved by the FDA for metastatic melanoma treatment and G47∆ which received conditional approval for the treatment of GBM in Japan. In prior work, our group has demonstrated that PTENα expression by an oHSV (HSV-P10) results in improved long-term survivors in intracranial tumor-bearing mice compared to HSVQ treatment. We aim to elucidate the mechanism of improved therapeutic efficacy of HSV-P10 against GBM and evaluate if HSV-P10 may overcome radio-resistance. RNA sequencing and GSEA analysis of primary human GBM cells infected with control HSVQ or HSV-P10 reveals that while HSVQ virus infection leads to an increase in genes regulating IL6-STAT3 pathway, pivotal in maintaining stemness properties, HSV-P10 infection leads to a reduction. HSV-P10 reduces CD133+/CD44+ stem cells, induces DNA damage and sensitizes the GBM cells to irradiation. Our findings reveal a novel mechanism induced by HSV-P10 in combination with irradiation where HSV-P10 modulates IL6-STAT3 signaling downregulating genes associated with stemness (Nestin, Sox2). HSV-P10 infection in combination with irradiation reduces GSC tumor sphere formation in vitro and sensitizes GBMs to radiotherapy in an intracranial mouse xenograft model. Our findings uncover a possible mechanism to overcome GSC-mediated therapy resistance to improve the therapeutic efficacy for GBM.
BIOM-22. BMX EXPRESSION LEVEL IN GLIOBLASTOMA PREDICTS HYPERSENSITIVITY TO IBRUTINIB TREATMENT
Authors: Sajina Shakya, Swetha Sundar, Violette Recinos, Christopher Hubert
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma (GBM) is the most prevalent and aggressive form of primary brain cancer and is notoriously drug and radiation resistant. The tumor recurrence and therapeutic resistance typically seen in GBM are largely driven by a small population of self-renewing, highly tumorigenic cells termed cancer stem cells (CSCs). The stem-cell-like properties of GBM CSCs are promoted, at least in part, by abnormal activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway. This activation can occur through aberrant expression of bone marrow and X-linked (BMX) protein, a non-receptor tyrosine kinase that is upregulated in glioma stem cells and may be targeted by the drug ibrutinib, currently undergoing clinical trials in GBM. In contrast, BMX is not expressed in normal non-cancerous stem cells of the brain. We discovered a striking dissimilarity where a subset of our patient-derived GBM specimens are up to 100-fold more sensitive to ibrutinib than others. We investigated the mechanisms behind this hypersensitivity by analyzing the expression levels of JAK/STAT3 signaling proteins. Here we discovered that the baseline expression value of BMX prior to treatment has a strikingly high predictive value for later later response to ibrutinib, primarily explaining the variation in a specimen’s ibrutinib sensitivity. Furthermore, we found that pediatric GBM tumor models are typically BMX-low and most likely to be hypersensitive to ibrutinib compared to their adult counterparts. Ibrutinib further suppresses GBM and DIPG proliferation in CSC-rich proliferative regions of organoid models. Our data suggest the potential use of BMX as a predictive biomarker of ibrutinib sensitivity, and provide an impetus for mechanistic and preclinical investigation of ibrutinib sensitivity, particularly in pediatric disease.
EXTH-27. TRANSLATABLE CCL3 THERAPY TO PROMOTE DC VACCINE MIGRATION AND EFFICACY AGAINST GLIOMA
Authors: Kelly Hotchkiss, Dahlia Halabi, Kristen Batich, Sarah Cook, Pam Norberg, Gary Archer, John Sampson
Journal: Neuro-Oncology (2022)

Abstract INTRODUCTION Prior evidence from our studies have identified chemokine ligand 3 (CCL3) as a key modulator to increase DC vaccine migration to vaccine draining lymph nodes (VDLNs) which is associated with improved long-term survival clinically. METHODS The effect of CCL3 treatment on DC vaccine migration ex vivo and in vivo (n=5), antigen-specific T cell responses (n=5), and efficacy against orthotopic GL261-OVA and SMA560 tumors (n=10) was studied in C57Bl/6 and VMdK mice. CCL3 treatment of mouse and human DC vaccines ex vivo was also tested in transwell assays. DCs were electroporated with OVA-mRNA or pulsed with ODC1 neoantigen peptide. DC migration and T cell responses were quantified by flow cytometry. Median overall survival (mOS) was measure in days (d) post-intracranial implantation. RESULTS Ex-vivo treatment of DC vaccines with 20ng/mL CCL3 (18 and 1hr pre-injection) increased transwell (mouse and human) migration and to VDLN. In vivo systemic CCL3 treatment also resulted in a dose-dependent increase in migration of DC vaccine to VDLN (10µg, 20µg, 50µg). CCL3 added to DC vaccination generated more tumor antigen-specific CD8+IFNγ+ T cells at 7-days. CCL3 with tumor antigen-DC treatment also resulted in significantly greater survival compared to OVA-DC alone (GL261-OVA: mOS DCvac: 19.5d, CCL3+DCvac 37d, p=0.0174; SMA560: mOS: DCvac: 25d, CCL3+DCvac: 48d, p=0.002). CONCLUSIONS These data combined with previous success of our DC vaccine clinical trials reflect the potency of CCL3 to enhance DC vaccine-specific migration, immune responses, and survival. CCL3 is a novel and safe adjuvant to overcome prior limitations in DC vaccine therapy and may be translatable to increase heterogeneous tumor antigen presentation following vaccine-targeted tumor killing. CCL3 used as a drug could increase vaccine migration without potential systemic toxicity in patients with glioblastoma.
MODL-04. ESTABLISHMENT OF A RELIABLE GLIOBLASTOMA TUMOR-ORGANOID PLATFORM ENABLES AUTOMATED HIGH-THROUGHPUT DRUG SCREENING ADVANCING PERSONALIZED MEDICINE
Authors: Christel Herold-Mende, Adrian Paul, Junguo Cao, Rolf Warta, Unterberg Andreas, Amir Abdollahi, Gerhard Jungwirth
Journal: Neuro-Oncology (2022)

Abstract Patient-derived tumor organoids (TOs) generated from tumor tissues within a few days are promising new models since under optimal conditions they preserve genotype, phenotype, the tumor microenvironment and the cellular heterogeneity of the parental tumor. This exciting new technology could provide representative avatars of the patient tumor in large numbers and thus can be exploited for drug discovery and drug screening purposes more efficiently than conventional cell lines or xenografted mouse models. We established standardized TOs from fresh glioblastoma (GBM) tissue after enzymatic dissociation into single cell suspensions and seeding cells into anti-adhesive 384-well plates. Over 1000 standardized TOs were generated per patient with a success rate of over 90%. Morphologically, TOs formed after two days. Size was stable over 10 days, with a slow outgrowth in some cases. A steady increase of the ATP signal over time indicated proliferative activity in TOs. Immunofluorescent stainings for tumor cells (GFAP), immune cells (macrophages and T-cells), and extracellular matrix (Tensascin C) confirmed a strong resemblance of TOs to their corresponding parental tumor, which was further substantiated by RNA-seq and DNA-methylation. Next, automated robot-based high-throughput drug screening (aHTS) on TOs was performed using a drug library of 166 FDA-approved antineoplastic drugs. Cell viability was assessed by CellTiter-Glo3D. In total, aHTS on TOs from 11 GBM patients including two recurrent GBMs showed a strong heterogeneity of drug responses. The top ten best-performing drugs consisted of 35 compounds targeting 14 different modes of action. Proteasome and HDAC-inhibitors demonstrated the highest efficacy. Respective targets were validated by knockdown experiments in GBM cells. Altogether, our data corroborate the future use of standardized GBM TOs as patient avatars for personalized drug testing by utilizing an automated high-throughput drug screening platform to better account for inter-patient-individual drug responses.
EXTH-04. PATIENT-DERIVED CELLS FOR EX VIVO DRUG SCREENING STUDIES OF GLIOMAS
Authors: Amin El-Heliebi, Tadeja Urbanic-Purkart, Kariem Mahdy-Ali, Christina Skofler, Lisa Gerlitz, Stefanie Stanzer, Joakim Franz, Nora Harbusch, Tobias Madl, Georg Widhalm, Karl Rössler, Martina Tomberger, Karin Mattersdorfer, Thomas Kroneis, Monika Oberhuber, Thomas Pieber, Barbara Prietl
Journal: Neuro-Oncology (2022)

Abstract BACKGROUND In precision oncology ex vivo drug screening systems have the potential to improve clinical outcomes. Traditionally, cancer drugs are tested on cancer cell line models, but these cannot represent an individual patient and are biologically too distinct. Drug screening systems usually rely on viability assays and correlations to genomic alterations. Beside genomic alterations, the cellular metabolism is significantly altered which may lead to drug resistance. Here we aim to establish a drug screening platform using tumor cells derived directly from the individual patient glial tumor tissue, create patient derived tumor cells (PDCs) and combine the outcomes from standardized viability- and genetic-assays with a new developed metabolomics platform. Materials and METHODS Fresh native tissue from patients harbouring low- and high-grade glioma are collected (n=46). Tumor tissue used for NMR-based metabolomic analyses and targeted sequencing analyses as well as PDC isolation. To preserve the original tumor similarity, tissue is short term cultured for two weeks, and PDCs are seeded and treated with a panel of clinical- and preclinical drugs followed by viability assessment, sequencing and metabolomic profiling. RESULTS Culturing of PDCs is successful in ≥ 85% of patient cases, provided that at least 2 g of tumor tissue is available. The automatized high throughput ex vivo drug response identifies drug candidates, which might become relevant for therapeutic approaches in future. It is possible to distinguish between IDH1-wild-type and IDH1-mutant tumors based on the metabolomic profile, which is confirmed by immunohistochemical staining and molecular analysis of IDH1 R132H-mutation. Strong metabolomic variations have been identified, including GABA, lactate, and myo-inositol levels between tumor and healthy tissue. CONCLUSION Entangling drug screening and genetic assays with metabolomic profiling of glial tumors enriches the information about cellular drug response and paves the way for future clinical studies and better understanding of underlying drug resistance mechanisms in gliomas.
BIOM-16. A MULTI-OMIC, FUNCTIONAL PRECISION ONCOLOGY METHOD TO IDENTIFY RESPONSIVE GLIOBLASTOMA TUMOR CELLS AT SINGLE CELL RESOLUTION
Authors: Yanti De Visser, Dena Panovska, Basiel Cole, Lotte Hermans, Asier Antoranz Martinez, Pouya Nazari, Maaike Van Trimpont, Marleen Derweduwe, Annelies Claeys, Steven De Vleeschouwer, Lien Solie, Paul Clement, Raf Sciot, Michiel Verfaillie, Thomas Daenekindt, Sven Bamps, Pieter Van Vlierberghe, Frederik De Smet
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma remains a highly malignant and intrinsically resistant brain tumor. Despite intensive research through which numerous potential druggable targets were identified, virtually all clinical trials of the past 20 years failed to improve the outcome for the vast majority of GBM patients. However, the identification of small subgroups of patients that showed an exceptional response across several trials, implies that, when selected more carefully, some GBM patients could probably still benefit from these therapies. Identifying these patients requires that suitable biomarkers are identified. In this project, we reassessed the molecular mechanisms of ten actionable compounds (selected from previously failed trials but for which exceptional responders had been observed) in a set of carefully selected patient-derived cell lines that were sensitive/resistant to the selected therapies. Moreover, to deal with tumor heterogeneity, we used a multi-omic functional precision oncology approach, combining scRNA-seq and CyTOF, to identify drug-specific biomarkers by comparing control and treated samples at single-cell resolution. By subsequently correlating the molecular signatures to eventual cytotoxicity profiles, we could identify intrinsically responsive tumor cells at the single-cell level within hours following drug exposure. Overall, this work lays the foundation for an actionable functional diagnostic assay that could help to identify eligible GBM patients in future clinical trials.
QLTI-16. ENHANCED RECOVERY AFTER LASER ABLATION SURGERY: A PRELIMINARY ANALYSIS OF A NOVEL PROGRAM
Authors: Martin Merenzon, Adam Levy, Tiffany Eatz, Alexis Morell, Dominique Higgins, Nitesh Patel, Michael Kader, Daniel Eichberg, Ashish Shah, Michael Silva, Victor Lu, Evan Luther, Marc Bloom, Ricardo Komotar, Michael Ivan
Journal: Neuro-Oncology (2022)

Abstract INTRODUCTION The concept of enhanced recovery after surgery (ERAS) due to standardized interventions has been gaining more relevance within neurosurgery. Advances were made both in protocols for spine and cranial surgery. These experiences described many benefits such as less psychological stress, reduction in hospitalization days, and lower hospital costs, without increasing the incidence of complications. However, no experience has described to date the applicability of an ERAS program for laser ablation thermal therapy (LITT). OBJECTIVE To describe our initial experience with the first enhanced recovery program reported for laser ablation for brain tumors. Secondly, to summarize the perioperative clinical outcomes of ERAS applied to LITT. METHODS We performed a retrospective analysis of all adult patients who underwent LITT for oncological lesions from 2013 to 2021. A multidisciplinary program was created by protocolizing interventions carried out along the path of the patient's hospitalization. Each recommendation was individually assessed for its appropriateness for enhancing recovery and for its validity with a focused literature review process. RESULTS A total of 184 patients were included, with a mean age of 60.7 ± 13.5 years, 35% males. 167 tumors were located in the supratentorial compartment, and 17 were infratentorial; the mean tumor diameter was 1.84 ± 1.04 cm. Among the pathologies treated 50.0% were metastasis, and 36.9% were glioblastomas. The mean postoperative day discharge was 1.2 ± 0.8 days. The readmission rate due to surgical complications within 30 days of surgery was 2.7%. These readmission rates fall into what is expected according to published literature without an ERAS program and longer hospital admissions. One death was recorded in the perioperative period. CONCLUSION Clinical interventions that could constitute an ERAS program are feasible in laser ablation of brain tumors. This study could be useful as a preliminary framework for the development of future guidelines.
DNAR-06. LOSS OF HISTONE H3 THREONINE 45 PHOSPHORYLATION DECREASES H3K36ME3 TO ABROGATE THE RADIATION-INDUCED DNA DAMAGE IN GLIOBLASTOMA MULTIFORME
Authors: Kiran Parwani, Jennifer Spangle, Leon McSwain, Ramona Haji Seyed Javadi, Anna Kenney, David Yu
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma multiforme (GBM) is the most common and fatal adult brain tumour. As radioresistance is a common occurrence and contributes to poor patient prognosis, exploiting the mechanistic basis of GBM radioresistance to develop new therapeutic approaches remains an unmet clinical need. Chromatin post-translational modifications (PTMs) can augment cancer radioresistance. Several chromatin PTMs are regulated by PI3K/AKT, which is frequently hyperactivated in GBM. Previous research demonstrated that the PI3K effector AKT phosphorylates histone H3 at threonine 45 (pH3T45) in response to DNA damage. To genetically dissect the function of H3T45 phosphorylation in the DNA damage response, we engineered the PI3K pathway-activated U87MG GBM cell line to stably overexpress wildtype H3, or a phospho-null H3T45A mutant. We show that in both the presence and absence of irradiation, cells harbouring the phospho-null H3T45A mutant decreases H3K36me3 on free and nucleosome-incorporated histones, which may suggest a deficiency in DNA repair protein recruitment. By surveying irradiation-induced 53BP1 foci formation, we demonstrate that loss of pH3T45 compromises damage repair through NHEJ by delaying the onset of repair signaling. We also observe a defect in DNA damage resolution, which enhances apoptosis to reduce cell clonogenicity and proliferative capacity. Irradiation does not further reduce clonogenicity and proliferation in the phospho-null H3T45A-expressing cells. Taken together, these data suggest a critical function for PI3K-directed H3T45 phosphorylation in DNA damage repair and that loss of pH3T45 may contribute to GBM radioresistance. We anticipate that further characterization of this phosphorylation event will inform the understanding of GBM radioresistance and may suggest novel therapeutic strategies.
TMIC-83. SEX AND ANTI-GLIOMA IMMUNITY
Authors: Bozena Kaminska, Natalia Ochocka, Pawel Segit, Kamil Wojnicki, Karol Jacek, Wieslawa Grajkowska
Journal: Neuro-Oncology (2022)

Abstract Glioblastomas (GBMs) are aggressive, inevitably lethal brain tumors that are massively infiltrated by myeloid cells supporting tumor growth. The main myeloid cell populations encompass brain-resident microglia, bone marrow (BM)-derived monocytes/macrophages and dendritic cells. Interestingly, patients with glioblastoma show sex-dependent differences in the incidence rate (male-to-female ratio of 1.6:1 in GBMs and 2:1 in most malignant mesenchymal GBMs), transcriptomic profiles and patient responses to a standard therapy. Sex-related differences such as higher expression of pro-inflammatory genes in female microglia and stronger upregulation of MHCII coding genes in microglia from male GL261-bearing mice were detected (Ochocka et al. 2021). We employed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) to reliably dissect myeloid cell identities, states and evaluate dynamics of myeloid infiltration during progression of murine GL261 gliomas. We demonstrate the diversity of myeloid cells within the glioma microenvironment: glioma-activated microglia are the major source of cytokines attracting other immune cells, whereas BM-derived cells show the monocyte-to-macrophage transition and immunosuppressive phenotypes. This transition is coupled with a phenotypic switch from the IFN-related to antigen-presentation and tumor-supportive signatures. Moreover, we found striking sex-dependent differences in transcriptional programs and composition of myeloid cells in gliomas. Higher abundance of protumor macrophages in males correlated with greater tumor size. Re-analysis of single-cell omics data from human GBMs revealed the predominance of inflammatory monocytes in female GBMs and abundance of protumor macrophages in male GBMs. Our findings expand understanding of the complexity of anti-tumor immune responses in gliomas and may guide future therapies in consideration of patient sex. Studies supported by National Science Centre Poland research grants PRELUDIUM16 2018/31/N/NZ3/01696, OPUS 14 2017/27/B/NZ3/01605 and 2020/39/B/NZ4/02683.